Camptothecin (CPT) is a potent DNA Topoisomerase I inhibitor with anti-tumor activity in hematological and solid tumors. However, it did not reach clinical use because of its poor solubility and high degrability. β-Cyclodextrin nanosponge (CN) have been demonstrated to be able to increase the solubility of lipophilic compounds and to protect them from degradation. In the present study, we evaluated whether β-Cyclodextrin nanosponge carriers can overcome CPT chemical disadvantages and improve the in vitro anti-tumor efficacy in the androgen refractory models of prostate cancer DU145 and PC-3 and the androgen sensitive model LNCaP. Camptothecin-loaded β-Cyclodextrin nanosponge (CN-CPT) showed sizes of about 400 nm, spherical shape and a drug loading of 38%. HPLC analysis, performed on the cell pellet after treatment with CN-CPT revealed that CPT concentration increased over time indicating a prolonged release of the drug. Moreover, CN-CPT inhibited Topoisomerase I activity, and induced DNA damage, and cell cycle arrest more effectively than CPT, indicating that the CN-CPT formulation does not affect activity of the drug. Moreover, Annexin V/Propidium Iodide staining showed an induction of cell death at low concentrations that were not effective for CTP. LNCaP cells were less sensitive to CPT than PC-3 and DU145 cells, but CN-CPT still exerted higher anti-proliferative activity and DNA damage ability than CPT. The experiments performed in LNCaP cells demonstrated that CN-CPT treatment inhibited expression of the androgen receptor at doses where CPT was ineffective. Our results demonstrated the higher anti-tumor effectiveness of CN-CPT compare to CPT in prostate cancer cells, supporting the relevance of future studies for the use of the β-Cyclodextrin nanosponge to deliver anticancer drugs in vivo.